Alternative pathway fof bovine complement Immunochemical studies on factor B-like serum protein and its conversion product B gamma 2.

Rabbits produced antibodies to a factor B-like serum protein (factor Bbov), its conversion product B gamma 2 and some other bovine serum proteins after repeated immunization with zymosan which previously had been incubated with fresh bovine serum. Such antisera were used to monitor purification of B gamma 2 from fresh bovine sera incubated with zxymosan. Subsequently, antisera specific for factor Bbov and B gamma 2 were produced. Antiserum produced against B gamma 2 cross-reacted with factor Bbov. Functional assays for factor Bbov were carried out in a hemolytic system with guinea pig erythrocytes in EGTA buffer. Heat inactivation (56 degrees C/5 min) of bovine serum destroyed the antigenicity of factor Bbov but not that of B gamma 2. Factor Bbov had an apparent molecular weight of 95,000 and B gamma 2 a molecular weight of 40,000 daltons. Conversion of factor Bbov to B gamma 2 was determined qualitatively by immunoelectrophoresis and quantitatively by radial immunodiffusion. Conversion of factor Bbov to B gamma 2 in bovine serum, in the presence of zymosan or cobra venom factor (CoVF) required Mg++ but not Ca++, did not occur in heat inactivated (56 degrees C/5 min) serum and was maximal, but not complete, when fresh bovine serum was incubated with zymosan (20 mg/mL) at 37 for two hours.     

Classical and alternate complement pathway activities in paired dairy cow-newborn calf sera

Hemolytic assays were used to compare alternate and classical C pathway activities in sera obtained from clinically normal newborn dairy calves and their mothers at the time of delivery. Mean alternate and classical CH50 concentrations in sera from newborn calves were both significantly lower than in their dams (P less than 0.001). The titer of alternate C pathway activity, expressed as CH50 units/ml, in sera from 17 calves was 12.9 +/- 5.5, whereas for the cows it was 25.8 +/- 6.2. The ratio of cow: calf serum alternate CH50 titers averaged 2.25 +/- 0.80 and ranged from 0.88 to 4.14. Classical CH50 titers were 78.0 +/- 42.7 units/ml in calf sera and 246.0 +/- 44.5 in cow sera. The ratio of cow: calf serum classical CH50 titers averaged 3.71 +/- 1.49 and ranged from 1.19 to 6.87. The wide range of values, noted for both the alternate and classical C pathways, within maternal and neonatal groups was assumed to reflect the biologic variability of complement levels in bovine serum. The possible relationships between deficient levels of alternate and classical CH50 activity in newborn calves and their susceptibility to infections is discussed.     

Defences of the bovine mammary gland against infection and prospects for their enhancement

The equine alternative complement pathway has been partially characterized and compared to the equine classical activation pathway. A dose-dependent lysis of RbRBC was observed with peak lytic values noted within 10 minutes at 37°C when rabbit red blood cells (RbRBC) were used as an alternative pathway activator. Sheep red blood cells (SRBC) sensitized with rabbit hemolysin or partially purified equine IgM antibodies were equally sensitive to lysis. Dilution of the commercial hemolysin by $\text{1}\text{5}$ reduced lysis from 90% to 38% in the presence of constant cell numbers. Hemolysis of SRBC peaked at 10 minutes and the majority of lysis occurred within 10 minutes. Dilution of equine sera by as little as $\text{1}\text{5}$ decreased hemolytic activity for SRBC to 21.5% from greater than 90% with undiluted sera. The alternative pathway protein, equine factor B, was tested using RbRBC and monitored by its differential susceptibility to heat treatment at 50°C. This treatment led to almost complete inactivation after a 15-minute incubation. An apparent heat-dependent decay of certain classical pathway components was also observed after 50°C treatment. This sensitivity was indicated by a reduction in the lytic activity for sensitized SRBC. Treatment for 15 minutes at 56°C with either RbRBC or SRBC was sufficient to abolish hemolytic activity in all equine sera tested. Chelation of cations with 0.04 M EDTA blocked expression of alternative and classical pathway activation; however, chelation of Ca⁺⁺ ions with 10 mM EGTA containing 1 mM Mg⁺⁺ ions permitted lysis of the RbRBC but not the SRBC. A dose-related Mg⁺⁺-ion dependence for RbRBC hemolytic activity was observed as the concentration of Mg⁺⁺ was increased to 1.0 mM. In addition, our results obtained with pre-colostral foal serum strongly suggest that natural antibody to RbRBC was of little importance in the lysis observed with these cells. These results also show that the equine alternative pathway activation may require Ca⁺⁺ ions. If Ca⁺⁺ ions are required, the equine alternative pathway is quite different from any other mammalian complement system so far described. Our results suggest that the alternative pathway of activation is of major importance in the equine complement system. Confirmation of this hypothesis requires both purification of the components involved as well as further characterization.     

Isolation, characterization, and quantitative analysis of ceruloplasmin from horses.

Ceruloplasmin (Cp) was isolated from fresh equine plasma by precipitation, cellulose chromatography, and improved ion-exchange chromatography. Purified equine Cp is a glycoprotein having a molecular weight of approximately 115,000. In electrophoresis, equine Cp migrated to the alpha 1-globulin region, its isoelectric point was about 4.15 and consisted of about 890 amino acid residues. Serum Cp concentration was measured by use of the single radial immunodiffusion method. In clinically normal horses, the mean (+/- SD) serum Cp concentration of newborn foals was 2.87 +/- 0.40 mg/ml and that of 3-month-old foals was 5.02 +/- 0.92 mg/ml, which was similar to the adult value. It reached a peak of 6.06 +/- 0.74 mg/ml in 2-year-old horses. The Cp concentration in mares was not statistically different for the perinatal period, but it decreased immediately before and after delivery. Concentration of Cp increased at 6 days after IM administration of turpentine oil, castration, or jejunojejunostomy in adult horses, and increased to peak values twice as high as baseline values at 7 to 14 days, returning to baseline values at 28 days after treatment. We concluded that equine serum Cp is an acute-phase reactive protein increased in the intermediary or later phase of acute inflammation.     

Determining the IgG concentrations in bovine colostrum and calf sera with a novel enzymatic assay

Background: Immune protection in newborn calves relies on a combination of the timing,volume and quality of colostrum consumed by the calf after birth.Poor quality colostrum with inadequate immunoglobulin concentration contributes to failed transfer of passive immunity in calves,leading to higher calf morbidity and mortality.Therefore,estimating colostrum quality and ensuring the transfer of passive immunity on farm is of critical importance.Currently,there are no on-farm tools that directly measure immunoglobulin content in colostrum or serum.The aim of this study was to apply a novel molecular assay,split trehalase immunoglobulin G assay(STIGA),to directly estimate immunoglobulin content in dairy and beef colostrum and calf sera,and to examine its potential to be developed as on-farm test.The STIGA is based on a split version of trehalase TreA,an enzyme that converts trehalose into glucose,enabling the use of a common glucometer for signal detection.In a first study,60 dairy and64 beef colostrum and 83 dairy and 84 beef calf sera samples were tested with STIGA,and the resulting glucose production was measured and compared with radial immunodiffusion,the standard method for measuring immunoglobulin concentrations.Results: Pearson correlation coefficients between the methods were determined and the sensitivity,specificity,and accuracy of the test were calculated for different colostrum quality and failed transfer of passive immunity cut-off points.The correlations of the STIGA measured by colorimetric enzymatic reaction compared to radial immunodiffusion for dairy and beef colostrum were 0.72 and 0.73,respectively,whereas the correlations for dairy and beef sera were 0.9 and 0.85,respectively.Next,STIGA was tested in a blinded study with fresh colostrum and serum samples where the correlation coefficient was 0.93 and 0.94,respectively.Furthermore,the performance of STIGA followed by glucometer readings resulted in correlations with radial immunodiffusion of 0.7 and 0.85 for dairy and beef colostrum and 0.94 and 0.83 for dairy and beef calf serum.Conclusions: A split TreA assay was validated for measurement of the immunoglobulin content of colostrum and calf sera using both a lab-based format and in a more user-friendly format compatible with on-farm testing.     

Equine haptoglobin: isolation, characterization, and the effects of ageing, delivery and inflammation on its serum concentration.

Haptoglobin (Hp) was isolated from equine serum by ammonium sulphate precipitation, anion-exchange chromatography and gel filtration. Equine Hp which migrated to the alpha 2-globulin region in electrophoresis, contained 2 fractions with molecular weights (NW) of 108,000 and 105,000, and each fraction consisting of 2 subunits. Quantitative measurement of Hp in equine serum was performed by the single radial immunodiffusion method using anti-equine Hp serum. In clinically normal horses, the highest concentration of serum Hp was found in newborn foals and a high value was maintained until 12 months of age. The concentration then decreased with age. Normal Hp values were 5.25 +/- 2.36 mg/ml in foals (less than or equal to 12 months old), 2.19 +/- 1.54 mg/ml in adult horses (greater than or equal to 18 months old) and 3.62 +/- 0.81 mg/ml in all horses. Serum Hp concentration in mares during the perinatal period in comparison with the normal adult female was high for 4 months pre-partum, a passing increase at delivery, and then decreased at 2 weeks post-partum returning to normal within 1 month of delivery. In horses with experimentally-induced inflammation, serum Hp concentration began to increase immediately after treatment and reached the highest value, 1.5 to 9 times higher than those of pre-treatment at 2 to 5 days, then decreased within 4 weeks. It was also elevated in most cases of horses with clinically inflammatory signs.     

Isolation, characterization and quantitation of canine alpha-1-acid glycoprotein

The present study was designed to investigate the physicochemical properties of canine alpha-1-acid glycoprotein (AGP), and to establish a method for measuring serum AGP in canine. Fifty-three normal beagles were used in the study. AGP was purified from normal canine sera by successive ammonium sulfate precipitation, ion-exchange chromatography and gel filtration. The serum AGP concentration was measured by single radial immunodiffusion (SRID). Canine AGP had a molecular weight of 42,000 +/- 2,000 Da and contained 40.9% carbohydrate. Gel isoelectric focusing revealed microheterogeneity with 6-7 bands in a pI range (isoelectric points) of 3.2-3.8. AGP migrated to the alpha(1)-globulin region on immunoelectrophoresis. The serum AGP level of 35 normal beagles was 283.4 +/- 113.3 mug/ml. Canine AGP was isolated, and its physicochemical properties were clarified. SRID may be a useful method for quantification of serum AGP in canine.     

Genetic and environmental factors affecting immunoglobulin G1 concentrations in ewe colostrum and lamb serum

Presuckle colostral samples and lamb serum samples taken 36 h postpartum were assayed for immunoglobulin G1 (IgG1) concentration (mg/ml) using single radial immunodiffusion. Breeds sampled included Polypay (P), Rambouillet (R), Targhee (T), Columbia (C), Finnish Landrace (F) and Finn crosses (Fx). Sources of variation examined in IgG1 concentration in colostrum (dam trait) included dam's sire breed, dam's sire, age of ewe and number of lambs born. All sources of variation were statistically significant. Least-squares means of IgG1 levels for sire breed were 80, 64, 67, 64, 72 and 69 mg/ml for P, R, T, C, F and Fx breed groups, respectively. A fetal stimulus may exist to increase the mass of IgG1 in colostrum available for multiple births (61, 69 and 77 mg/ml for single, twin and triplet, respectively). Ewe age was a significant source of variation because of a high mean concentration of IgG1 in the yearling's colostrum (100 mg/ml), whereas only slight differences occurred between the other age groups (65 to 67 mg/ml), except for the 7-yr older group (53 mg/ml). Sources of variation examined in IgG1 concentration of lamb serum at 36 h postpartum (lamb trait) included lamb's sire breed, lamb's sire, age of dam, birth type and sex, with dam's colostral IgG1 concentration and day born as covariates. Sire within breed, birth type and the two covariates were significant. Least squares means for sire breed were 36, 32, 33, 32, 31 and 32 mg/ml of serum for P, R, T, C, F and Fx groups, respectively. Lamb serum IgG1 decreased as birth type increased. The heritability of IgG1, estimated by paternal half-sib analyses, was .19 +/- .12 for colostrum and .18 +/- .06 for lamb serum.     

Use of Fourier-transform infrared spectroscopy for the diagnosis of failure of transfer of passive immunity and measurement of immunoglobulin concentrations in horses

BACKGROUND: The economic, accurate, and rapid screening of foals for failure of transfer of passive immunity (FPT) is essential to ensure timely intervention. HYPOTHESIS: Infrared (IR) spectroscopy of foal sera and pattern recognition may be used to diagnose FPT and quantify serum IgG. SAMPLES: Sera from 194 foals (24-72 hours) with serum immunoglobulin G (IgG) concentrations determined previously by radial immunodiffusion assay (RID) were used. METHODS: IR spectra were recorded for the serum samples, and the data were randomly divided into training and independent test sets, each containing both FPT-positive (IgG <400 mg/dL) and non-FPT samples. A genetic optimal region selection algorithm and linear discriminant analysis were used to partition the training spectra, and the resulting classifier was then validated by comparing the IR-predicted FPT status for each of the test samples to that provided by the RID IgG assay. A quantitative IR-based assay for IgG was developed using partial least squares (PLS) and validated by testing its ability to predict IgG concentrations. RESULTS: Specificity, sensitivity, and accuracy for the combined data were 92.5, 96.8, and 95.9%, respectively. Corresponding positive (88.1%) and negative predictive (98.0%) values determined a success rate of 95-97% as compared to RID-based IgG concentrations. The IR-based quantitative assay yielded correlation coefficients for IR spectroscopy versus RID-based IgG concentrations of 0.90 and 0.86 for the training and test sets, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE: The overall performance of the IR-based test was similar to that of the colorimetric assay and was superior and more economic than other available tests.     

A radial immunodiffusion enzyme assay for detection of antibody to equine rhinopneumonitis virus (EHV-1) in horse serum

A radial immunodiffusion enzyme assay (RIDEA) was developed for detection and quantitation of antibodies to equine herpes virus-1 (EHV-1) in horse sera. The detection and quantitation of EHV-1 antibody levels were based on the diameter of the radial diffusion zone of specific antibody in each serum sample reacting with EHV-1 antigen. The circular zone was made visible using peroxidase-conjugated rabbit anti-horse immunoglobulin G and a substrate containing hydrogen peroxide. The results of the RIDEA were compared with those of virus neutralization (VN) and enzyme-linked immunosorbent assay (ELISA) and found to be highly correlated. The relative sensitivity and specificity (percentage of agreement with VN test) were found to be 98.2 and 92.5%, respectively. Because the test procedure is relatively easy to perform, the RIDEA could be used as a field test to detect antibodies to EHV-1 in horses.     

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.     

Isolation of the fifth component of the bovine complement system

Bovine C5 has been isolated from fresh bovine serum by a five-step procedure: polyethylene glycol precipitation, sequential ion-exchange chromatography on DEAE-Sephacel and CM-Sephadex, hydroxylapatite chromatography, and affinity chromatography. The purified C5 was a protein of apparent molecular weight 202,000 +/- 9,000 composed of two chains: an alpha-chain of molecular weight 127,000 +/- 5,000 and a beta-chain of molecular weight 74,000 +/- 2,000. The alpha-chain was cleaved by Sepharose-CVF.Bb (a cobra venom factor (CVF)-induced C3/C5 alternative pathway convertase) in the absence of any C3 or C3b. The monocarboxylic acid form of K-76, a sesquiterpene compound isolated from the culture filtrates of Stachybotris complementi, inhibited the alternative pathway of bovine serum, and the inhibited hemolytic activity was restored, in a dose dependent manner, by bovine C5. This provided the basis for a C5 functional assay throughout the purification procedure. The purified C5 showed species specificity and was functionally distinct from bovine C3.     

A simple hemolytic assay for bovine complement component C3

A simple, one-step, alternative pathway (AP) hemolytic assay for bovine C3 has been developed. Methylamine was used to prepare a bovine serum reagent, R3, functionally depleted of C3. The addition of purified bovine C3 to the R3 reconstituted, in a dose-dependent manner, the hemolytic activity for unsensitized heterologous erythrocytes. The assay was used to determine relative levels of C3 in different bovine serum samples. Human C3 and bovine C3 were interchangeable in the assay. Reconstitution of bovine and human R3 reagents with homologous or heterologous C3, in the presence of different species of erythrocytes, provided evidence that cell surface regulation of the homologous hemolytic AP may not be limited to the assembly and activity of the C3 convertase. The AP assay was more sensitive and less complex to perform than a standard classical pathway assay for bovine C3.     

Hemolytic assay for goat (caprine) and swine (porcine) complement

Optimal conditions for assaying hemolytic complement of goat (caprine) and swine (porcine) sera were determined. Effects of the following were tested: pH, ionic strength, calcium and magnesium ion concentrations, time and temperature of incubation, and ethylenediamine tetracetate concentration. Guinea pig erythrocytes sensitized with goat or cattle antibodies were the most sensitive target cells for goat complement. Sheep and cattle erythrocytes sensitized with rabbit hemolysin were the best target cells for swine complement. Barbital buffer, pH 7.3, ionic strength of 90 nmM relative salt concentration, containing 0.5 mM CaCl2 and 1 mM MgCl2 was the best for swine complement assay. Goat complement lysed best in a barbital buffer, pH 8, ionic strength of 90 to 120 mM of relative salt concentration, in presence of 0.5 mM CaCl2 and 1 mM MgCl2. The optimal incubation temperature was 37 degrees C for both complements. The complement dependent lysis required 75 minutes to reach its maximum. Ethylenediamine tetracetate in 4 mM concentration completely inhibited lysis by both species complements. The CH50 for goat sera varied between 18 and 75 per ml, in swine sera between 75 and 210 per ml.     

Isolation, characterization, and quantitative measurement of serum alpha 1-acid glycoprotein in cattle

Bovine alpha 1-acid glycoprotein (alpha 1AG) was purified from pooled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gel filtration. Bovine alpha 1AG had a molecular weight of 42,000 +/- 2,000 and a sedimentation coefficient of 3.4S. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the alpha 1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine alpha 1AG in serum. The mean serum value of alpha 1AG in 152 healthy Holstein cattle (1-12 years old) was 283.2 +/- 82.3 micrograms/ml. Elevated values (cut-off value = 450 micrograms/ml) were observed in cattle with traumatic pericarditis (100%), arthritis (100%), mastitis (91%), pneumonia (70%), and mesenteric liponecrosis (43%).     

Effects of various treatments of bovine complement on its lytic efficacy measured by two different tests

Bovine complement was treated with various agents known to activate or inactivate one or more of the cascade components. The treated complement was then assessed for remaining hemolytic activity by a tube titration test and a radial hemolysis method. Divalent cation chelators (EDTA and EGTA); immune complexes prepared with serum and IgM, IgG1, IgG2, and IgA isotypes; smooth and rough lipopolysaccharides and lipid A; hydrazine; zymosan; cobra venom factor and brown recluse spider venom caused depletion of complement as determined in the tube titration test. Immune complexes (prepared with serum); hydrazine; cobra venom factor; EDTA and smooth lipopolysaccharide caused loss of hemolytic activity in the radial hemolysis test. This evidence suggests that the radial hemolysis test assessed complement consumed by the alternate pathway, while the tube titration method measured classical pathway consumption.     

Genetics of immunocompetent traits in a synthetic broiler dam line

Three hundred and three chicks of both sexes, from a synthetic dam line (SDL) of broiler chickens, were studied for economic traits (body weights at 4, 5 and 6 weeks of age) and immunological traits (humoral and cell mediated immune responses, and serum lysozyme concentration). The objective was to evaluate these traits and to estimate their genetic and non-genetic parameters. The humoral immune response was assessed by estimating the antibody response to sheep red blood cells using the haemagglutination (HA) test and serum IgG concentration using single radial immunodiffusion (SRID). The cell mediated immune (CMI) response was estimated as in vivo response to a mitogen (PHA-P). Serum lysozyme was measured by lysoplate assay. Least squares means for body weight at 4, 5 and 6 weeks were 684 +/- 20, 920 +/- 19 and 1205 +/- 28 g, HA titre was 6.289 +/- 0.246, CMI was 0.438 +/- 0.015 mm, lysozyme was 1.860 +/- 0.047 microg/ml and IgG was 6.287 +/- 0.194 mg/ml. There was an effect of sire on HA titre and on body weight at 4, 5 and 6 weeks of age; males were heavier than females. Heritability estimates were high for body weights but low for immunological traits. Phenotypic correlations (rp) among body weights were high and positive but were very low between body weights and most immunological traits. Among the immunological traits all rp were very low. Genetic correlations (rg) of body weights were positive and medium to high with CMI and HA and negative with serum IgG.     

A rapid and simplified technique for the assay of chicken hemolytic complement.

The technique described is a modification of a qualitative hemolytic radial diffusion technique. The test involves the use of sensitized sheep erythrocytes that have been incorporated into agarose. Tube dilutions were made of chicken serum and samples of each dilution were placed into wells cut in the agarose. The test is quantitative for hemolytic complement in that the highest dilution showing visible hemolysis of sensitized erythrocytes in agarose is determined to be the endpoint for that serum sample. The test as compared with the standard tube assay was determined to be less sensitive by approximately one dilution. The advantages of speed, simplicity, and cost more than offset the decrease in sensitivity of the test.     

Interaction of turkey complement with Escherichia coli isolated from turkeys

The role of turkey complement in a serum bactericidal reaction was determined using serum-sensitive and serum-resistant Escherichia coli isolated from turkeys. Inactivation of complement by heating serum (56 C for 40 minutes) or by treating serum with 10 mM EDTA eliminated bactericidal activity. Serum-sensitive E coli organisms were killed by turkey serum treated with 10 mM ethylene glycol-bis-beta-(aminoethyl ether)-N,N,N',N'-tetraacetic acid and 5 mM MgCl2. Exposure of normal turkey serum to serum-sensitive or serum-resistant E coli resulted in equivalent reductions in hemolytic activity of serum. Treatment of serum-resistant E coli with antibody rendered the bacteria sensitive to bactericidal effects of normal turkey serum. Serum-sensitive E coli organisms were readily killed by an alternative complement pathway, serum-sensitive and serum-resistant E coli activated the complement system equally well, and antibody was required for complement-mediated killing of certain serum-resistant E coli organisms from turkeys.     

Humoral immunity in disseminated Aspergillus terreus infection in the dog

Aspects of humoral immunity were studied in 17 dogs with disseminated aspergillosis (16 cases Aspergillus terreus, 1 case Aspergillus flavipes). All dogs had markedly raised serum IgG levels by single radial immunodiffusion (range 1500-6000 mg dl-1). Despite this, serum antibody to A. terreus was demonstrated in only 7/16 cases by agar gel diffusion, 9/16 cases by counter immunoelectrophoresis, 10/16 by ELISA and 11/16 by an indirect immunofluorescence assay. Serum antibody was also detected in 2/5 clinically normal relatives of 2 cases, indicating previous exposure or subclinical infection.